Integrins are heterodimeric cell surface adhesion receptors which play an important role in normal and pathological processes including hemostasis and thrombosis. These receptors contain both an .alpha. and a .beta. subunit. The integrin dimer has a cytoplasmic domain associated with the cytoskeleton. Thus, integrins form a critical connection between the extracellular matrix and the internal structural elements of a cell.
The most abundant integrin on platelets is .alpha..sub.IIb .beta..sub.3. .alpha..sub.IIb and .beta..sub.3 are type I transmembrane glycoproteins that contain 1008 and 762 amino acids, respectively. These glycoproteins are complexed together in membranes as non-covalent heterodimers. More than 95% of each subunit is extracellular; each has a single transmembrane domain and a short cytoplasmic tail (20 amino acids in .alpha..sub.IIb and 47 amino acids in .beta..sub.3).
.alpha..sub.IIb .beta..sub.3 mediates platelet aggregation as well as platelet adhesion and spreading on extracellular matrices. The affinity of this integrin for its extracellular ligands, arginine-glycine-aspartic acid (RDG)-containing ligands such as fibrinogen, von Willebrand factor (vWf), fibronectin and vitronectin, is regulated by the cell by a process known as "inside-out" signaling. Binding of a ligand such as fibrinogen to .alpha..sub.IIb .beta..sub.3 initiates a cascade of signaling reactions necessary for full platelet adhesion. This process is referred to as "outside-in" signaling.
This regulation by the cell is an important feature of the ligand-receptor interaction. Platelets normally circulate in a resting state in which the apparent affinity of .alpha..sub.IIb .beta..sub.3 for ligands is low. When platelets enter a vascular wound or encounter a physiological agonist, such as thrombin or ADP, or an activator of protein kinase C, such as phorbol myristate acetate (PMA), affinity for the receptor is rapidly increased resulting in ligand binding and platelet aggregation. (Bennett, J. S. and Vilaire, G., J. Clin. Invest. 1979, 64, 1393). Platelet aggregation is initiated by the bridging of .alpha..sub.IIb .beta..sub.3 complexes on adjacent platelets by fibrinogen or vWf. This increase in affinity can be prevented or reversed by addition of compounds that result in increased amounts of intracellular cyclic AMP or cyclic GMP such as prostaglandin I.sub.2 or nitric oxide, respectively. (Graber, S. E. and Hawiger, J., J. Biol. Chem. 1982, 257, 14606).
Platelet activation induces a conformational change in .alpha..sub.IIb .beta..sub.3, exposing a ligand-binding site that is likely to be composed of several discontinuous regions in the amino-terminal portions of both the .alpha..sub.IIb and the .beta..sub.3 subunits. (Sims et al. J Biol Chem 1991, 266, 7345-7352; Plow et al. Semin Thromb Hemost 1992, 18, 324-332). Receptor affinity is believed to be regulated through cytoplasmic tails of the .alpha..sub.IIb .beta..sub.3. Substitution of the cytoplasmic domains of .alpha..sub.IIb .beta..sub.3 with those of .alpha..sub.5 .beta..sub.1 in Chinese hamster ovary cells conferred an energy-dependent high affinity state on the extracellular portions of .alpha..sub.IIb .beta..sub.3. (O'Toole et al. J Cell Biol 1994, 124, 1047-1059). It is believed that intracellular signals generated by an agonist or antagonist result in modifications in the cytoplasmic domain of the .alpha..sub.IIb and .beta..sub.3 subunits. These modifications lead to changes in the extracellular portion of the receptor that increase or decrease accessibility of the ligand binding site. (Ginsberg et al. Curr. Opin. Cell. Biol. 1992, 4, 766). The study of recombinant forms of .alpha..sub.IIb .beta..sub.3 have shown that the affinity state of the fibrinogen receptor is influenced by sequences in the receptor's cytoplasmic tails in a cell type specific and energy-dependent manner. Thus, it has been suggested that specific cellular proteins interact with the tails and regulate receptor function. (O'Toole et al. J Cell Biol 1994, 124, 1047-1059).
Only a few intracellular proteins which interact with the cytoplasmic domains or tails of the .alpha..sub.II and .beta..sub.3 subunits and regulate inside-out and outside-in signaling have been identified. Otey et al.(J Cell Biol 1990, 111, 721-729) demonstrated that .alpha.-actinin binds to the cytoplasmic domain of the .beta. subunit of intact integrins from both the .beta..sub.1 and .beta..sub.3 subfamilies. They have also attempted to identify the binding site for .alpha.-actinin within the .beta..sub.1 integrin cytoplasmic domain. (Otey et al. J. Biol. Chem. 1993, 268, 21193-21197).
Human cDNAs have now been identified which express .beta..sub.3 integrin subunit specific polypeptides.